Synthesis and in vitro cytotoxicity of novel long chain busulphan analogues

Article abstract of DOI:10.1016/S0960-894X(01)00084-1

Two novel long chain alkanediol dimethanesulphonates, analogues of busulphan, were synthesized. Their in vitro cytotoxicity was evaluated against six solid tumor cell lines (A2780, H322, LL, WiDr, C26-10 and UMSCC-22B). 2-Tetradecylbutane-1,4-diol dimethanesulphonate was proved to be the most active compound exhibiting IC₅₀ values between 20.82 and 26.36 μM.

Full text of DOI:10.1016/S0960-894X(01)00084-1

Bioorganic & Medicinal Chemistry Letters 11 (2001) 861–863
Synthesis and In Vitro Cytotoxicity of Novel Long Chain
Busulphan Analogues
George Kokotos, * Violetta Constantinou-Kokotou, Jose M. Padro
´ ´
nc
a,
b
c
and Godefridus J. Peters
aLaboratory of Organic Chemistry, Department of Chemistry, University of Athens, Panepistimiopolis, Athens 15771, Greece
bChemical Laboratories, Agricultural University of Athens, Athens 11855, Greece
cDepartment of Medical Oncology, University Hospital VU, PO Box 7057, 1007 MB Amsterdam, The Netherlands
Received 21December 2000; revised 5 February 200 1; accepted 5 February 2001
Abstract—Two novel long chain alkanediol dimethanesulphonates, analogues of busulphan, were synthesized. Their in vitro cyto-
toxicity was evaluated against six solid tumor cell lines (A2780, H322, LL, WiDr, C26-10 and UMSCC-22B). 2-Tetradecylbutane-
1
#
,4-diol dimethanesulphonate was proved to be the most active compound exhibiting IC50 values between 20.82 and 26.36 mM.
2001Elsevier Science Ltd. All rights reserved.
7
Many chemotherapeutic agents used in the treatment of
cancer are bifunctional and are able to crosslink
biological macromolecules. Their most important cel-
lular target is believed to be DNA, and in the case of
alkylating agents the formation of interstrand crosslinks
may be the most relevant cytotoxic lesion. Crosslinking
starts by an initial covalent reaction of drug with an elec-
trophilic site on the DNA to form a monoadduct, fol-
lowed by a second reaction with the other DNA strand.
structural similarity to busulphan, was found to have
not only excellent antileukemic activity against P388
and L1210 mouse leukemia in vivo, but also good
1
6
activity against some solid tumors.
Alkanediol dimethanesulphonates of general formula
CH SO O(CH ) OSO CH constitute a very interesting
3
2
2 n
2
3
class of bifunctional chemotherapeutic agents. The best
known agent of this homologous series is busulphan
Recently we have been involved in the synthesis and
8
,9
(n=4) (1), which has been one of the drugs of choice in
the treatment of chronic myeloid leukemia for the last
study of novel lipophilic cytotoxic agents.
In this
paper the synthesis and in vitro cytotoxic activity of two
new long chain busulphan analogues is described. The
presence of the long chain confers lipophilicity to these
analogues that may facilitate transport across the cel-
lular membrane and increase intacellular drug accumu-
lation thus improving drug effectiveness. Since
structurally similar alkylating agents can differ in their
anticancer activity, we designed two analogues with
different relative positions of the two alkylating centers.
2
,3
3
0 years. It was suggested that busulphan produced
crosslinking in DNA through a GG bridge, but no dis-
tinction could be made as to whether this was derived
4
,5
from inter- or intrastrand crosslinks. It has also been
reported that busulphan induced DNA–protein cross-
links in human cell lines and mouse leukemia cell lines
6
after prolonged drug treatment.
Bifunctional alkylating agents, like busulphan analo-
gues, could be of great importance as new anticancer
agents. Hepsulfam (2), an anticancer agent with
Chemistry
Commercially available hexadecane-1,2-diol (3) was
chosen as starting material. Long chain diol 3 was con-
verted into the corresponding dimethanesulphonate 4
*Corresponding author. Fax: +30-1727-4761; e-mail: gkokotos@
cc.uoa.gr
0960-894X/01/$ - see front matter # 2001Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00084-1

862
G. Kokotos et al. / Bioorg. Med. Chem. Lett. 11 (2001) 861–863
3 2 4 4
Scheme 1. (i) MsCl, Et N; (ii) NaCN, DMF; (iii) H SO ; (iv) LiAlH .
Results and Discussion
by treatment with methanesulphonyl chloride in the
presence of triethylamine. Methanesulphonyloxy groups
were replaced by cyano groups by treatment with
sodium cyanide. The dinitrile 5 was hydrolyzed under
acidic conditions and the diacid 6 was reduced by
The long chain dimethanesulphonates 4 and 8 as well as
busulphan were tested for their cytotoxicity against six
cell lines of various origin. The in vitro experiments
were performed with the following cell lines: A2780, a
human ovarian cancer cell line; H322, a human non-
small cell lung cancer (NSCLC) cell line (subtype BAC,
NCI); LL, a murine NSCLC cell line; WiDr, a human
colon carcinoma cell line; C26-10, a murine colon car-
cinoma cell line; and UMSCC-22B, a human head and
LiAlH . 2-Tetradecylbutane-1,4-diol (7) was converted
4
into the target busulphan anologue 8. Busulphan was
also prepared by treatment of 1,4-butanediol with
methanesulphonyl chloride (Scheme 1).
All intermediates and final products gave satisfactory
analytical and spectroscopic data in full accord with
1
6
neck squamous cell carcinoma cell line. The cytotoxi-
city of the compounds is expressed as IC , which is the
1
0
their assigned structures.
5
0
drug concentration causing a 50% growth inhibition.
The IC50 values exhibited by the compounds are
summarized in Table 1.
In vitro cytotoxicity assay
Cell culture: cells were grown in monolayers in RPMI
1
640 supplemented with 5% heat inactivated fetal calf
ꢀ
Busulphan was proved inactive against all these solid
tumors. 2-Tetradecylbutane-1,4-diol dimethanesulphon-
ate (8) gave the best results against all the cell lines
exhibiting IC50 between 20.82 mM and 26.36mM. Hex-
adecane-1,2-diol dimethanesulphonate (4) exhibited
weaker activity (IC50 between 25.59 and 59.72 mM). In
the case of LL cell line the activity of compound 8 was
slightly higher than that of compound 4, and in WiDr
and C26-10 comparable. However, in the case of A2780,
H322 and 22B cell lines an approximate 2-fold increase
of the activity was observed.
serum and 2 mM l-glutamine in a 37 C, 5% CO , 95%
2
humidified air incubator. Exponentially growing cells
were trypsinized and resuspended in antibiotic contain-
ing medium (50 mg gentamicin/mL); single cell suspen-
sions displaying ꢁ97% viability by trypan blue dye
exclusion were subsequently counted. After counting,
dilutions were made to give the appropriate cell den-
sities for inoculation onto the microtiter plates. Cells
were inoculated in a volume of 100 mL per well at den-
sities of 5000 and 20,000 cells per well, based on their
1
1,12
doubling times.
It has been recognized that the relative position of the
two alkylating centers within bifunctional alkylating
agents is of importance with regard to their anticancer
Chemosensitivity testing: the assay was performed in
6-well plates using the National Cancer Institute pro-
9
1
3
tocol. Briefly, pure compounds were initially dissolved
in DMSO at 400 times the desired final maximum test
concentrations. Each agent was tested at five different
dilutions. Immediately after preparation of these inter-
mediate dilutions, 100 mL aliquots of each dilution were
added to the appropriate 96-well plate wells. Drug
incubation times were 72 h, after which cells were pre-
cipitated with 25 mL ice-cold 50% (w/v) trichloroacetic
acid and fixed for 60 min. Then the sulforhodamine B
Table 1. In vitro cytotoxicity of busulphan and analogues 4 and 8
a
Cell line
IC50 (mM)
Busulphan
4
8
A270
H322
LL
WiDr
C26-10
>100
>100
>100
45.45 (Æ13.37)
59.72 (Æ6.72)
28.43 (Æ5.88)
25.59 (Æ5.59)
30.97 (Æ4.92)
46.47 (Æ5.85)
25.89 (Æ1.51)
25.12 (Æ3.12)
20.82 (Æ0.64)
24.85 (Æ1.97)
26.26 (Æ4.22)
26.36 (Æ3.87)
>100
92.50 (Æ9.56)
1
4,15
(SRB) assay was performed. The optical density
OD) of each well was measured at 492 nm using a 96-
2
2B
>100
(
well plate reader (Titertek Multiscan MCC/340; Flow
Laboratories).
aValues are means of three experiments, standard deviation is given in
parentheses.

G. Kokotos et al. / Bioorg. Med. Chem. Lett. 11 (2001) 861–863
863
activity.17 The ability of bifunctional dimethanesulpho-
nates to span only selected target nucleophilic distances,
coupled with the availability and reactivity of these
sites, is crucial to the type and quantity of DNA adducts
8. Kokotos, G.; Theodorou, V.; Constantinou-Kokotou, V.;
Gibbons, W. A.; Roussakis, C. Bioorg. Med. Chem. Lett.
1
9
998, 8, 1525.
. Padron, J. M.; Martın, V. S.; Hadjipavlou-Litina, D.;
´
Noula, C.; Constantinou-Kokotou, V.; Peters, G. J.; Kokotos,
´
1
8,19
that may be produced.
The results obtained in this
G. Bioorg. Med. Chem. Lett. 1999, 9, 821.
study show that 1,4-distance between the alkylating
centers leads to better results in comparison with 1,2-
distance. Furthermore, both lipophilic derivatives 4 and
ꢀ
1
1
0. For example: compound 4: mp 52–54 C. H NMR (200
MHz, CDCl ) d ppm: 0.85 (t, 3H, J=7 Hz, CH ), 1.25 (m,
4H, 12ÂCH ), 1.70 (m, 2H, CH CHOSO ), 3.05 (s, 6H,
3
3
2
2
2
2
2
8
are more active than busulphan, indicating that the
ÂCH
3 2 2 2
OSO ), 4.30 (m, 2H, CH OSO ), 4.85 (m, 1H, CH).
presence of the long chain facilitates the molecular
interactions with DNA increasing the cytotoxic activity.
18 38 6 2
Analysis for C H O S (414.62): calcd C 52.14, H 9.24%.
ꢀ
1
Found C 52.28, H 9.37%. Compound 8: mp 53–55 C;
H
NMR (200 MHz, CDCl ) d ppm: 0.86 (t, 3H, CH ), 1.24 (m,
3
3
2
6H, 13ÂCH
2
), 1.88 (m, 3H, CH, CH
2
), 3.05 (s, 6H,
2
ÂCH OSO
3
2
2 38 6 2
), 4.20 (m, 4H, 2ÂCH ). Analysis for C18H O S
Acknowledgements
(
442.68): calcd C 54.26, H 9.56%. Found C 54.39, H 9.62%.
1. Bergman, A. M.; Ruiz van Haperen, V. W. T.; Veerman,
1
JMP acknowledges the Fundacio
La Palma, Islas Canarias, Spain) for a postdoctoral
grant.
´
n Dr. Manuel Morales
G.; Kuiper, C. M.; Peters, G. J. Clin. Cancer Res. 1996, 2, 521.
2. Van Moorsel, C. J. A.; Veerman, G.; Bergman, A. M.;
Guechev, A.; Vermoken, J. B.; Postmus, P. E.; Peters, G. J.
(
1
Seminars in Oncology 24 (Suppl. 7) 1997, 1 7.
1
3. Monks, A.; Scudeiro, D.; Skehan, P.; Shoemaker, R.;
Paull, K.; Vistica, D.; Hose, C.; Langley, J.; Cronise, P.; Vai-
gro-Wolf, A.; Gray-Goodrich, M.; Campbell, H.; Mayo, J.;
Boyd, M. J. Natl. Cancer Inst. 1991, 83, 757.
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